Development of a simple and versatile in vitro method for production, stimulation, and analysis of bioengineered muscle

In recent years, 3D in vitro modeling of human skeletal muscle has emerged as a subject of increasing interest, due to its applicability in basic studies or screening platforms. These models strive to recapitulate key features of muscle architecture and function, such as cell alignment, maturation, and contractility in response to different stimuli. To this end, it is required to culture cells in biomimetic hydrogels suspended between two anchors. Currently available protocols are often complex to produce, have a high rate of breakage, or are not adapted to imaging and stimulation. Therefore, we sought to develop a simplified and reliable protocol, which still enabled versatility in the study of muscle function. In our method, we have used human immortalized myoblasts cultured in a hydrogel composed of MatrigelTM and fibrinogen, to create muscle strips suspended between two VELCROTM anchors. The resulting muscle constructs show a differentiated phenotype and contractile activity in response to electrical, chemical and optical stimulation. This activity is analyzed by two alternative methods, namely contraction analysis and calcium analysis with Fluo-4 AM. In all, our protocol provides an optimized version of previously published methods, enabling individual imaging of muscle bundles and straightforward analysis of muscle response with standard image analysis software. This system provides a start-to-finish guide on how to produce, validate, stimulate, and analyze bioengineered muscle. This ensures that the system can be quickly established by researchers with varying degrees of expertise, while maintaining reliability and similarity to native muscle.


MATERIALS TEXT
White Velcro fabric (no adhesive backing). White color ensures that no dyes can leach into the culture. Follow BSL-2 guidelines for handling human cell lines.
Paraformaldehyde is a toxic chemical, handle in a fume hood and wear appropriate PPE (gloves, lab coat).
α-Bungarotoxin is a potent neurotoxin. At the used concentrations and in a standard manipulation setting, it is not considered a hazardous solution, but should be handled with care (gloves, avoid spills/skin exposure/ingestion).

Protocol overview:
General work ow of the protocol 1 Prepare VELCRO ® pieces. Only use white velcro without adhesive backing. Using only the "loop" (soft) part of the VELCRO ® , cut arrow-shaped velcro pieces according to directions in Figure 1. Use a pencil (not a marker) to divide the VELCRO ® into 12 x 4 mm strips, as shown in Figure 1. Then, cut these strips in half ( Figure 1A), which can be easily done by folding the strip and cutting along the fold. Next, cut the arrowheads as shown in Figure 1B.  Weigh 10 parts of elastomer and 1 part of curing agent in a plastic cup. Mix the components thoroughly with a glass rod, until the mixture is full of small bubbles and opaque in appearance.
Then, leave the mixture in a vacuum dessicator for 01:00:00 to completely remove bubbles.
PDMS glue can be prepared in bulk, and stored at -20 °C until the next preparation of muscle culture plates. When needed, it can be brought back to room temperature or brie y heated in a 37 °C water bath to reduce viscosity. This is convenient for reducing the total preparation time in subsequent batches of plates.
3 Mark a line of 1.2 cm in the center of each 35 mm ∅ Petri dish. In order to obtain consistently centered markings, it is recommended to make a small stencil out of paper or plastic ( Figure 2). If desired, the permanent marker lines can be erased with 70% ethanol after preparation to avoid capturing them in microscopy images.

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Attach VELCRO ® anchors to the Petri dishes, using the marked line as a guide. Use a small spatula to dab glue onto the underside of the VELCRO ® pieces, and then place them on the lines using tweezers, as shown in Figure 3. Make sure that the tips of the anchors are aligned, and the ends of the markings are aligned to the start of the arrowhead region of the anchors.

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Once the VELCRO ® pieces are attached, leave the plates at Room temperature Overnight to cure the PDMS glue. Then, store them until further use.

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Defrost myoblast cell line in a T75 ask and culture cells in their recommended maintenance medium. Human myoblasts must be passaged at a maximum of 60-70% con uence to avoid differentiation induced by cell-cell contacts.
In this section, all steps are performed inside a biological safety cabinet (vertical ow / BSL-2 preferrable). 7 Amplify cells to the needed amount. Take into account that around 3·10 6 cells are needed for each muscle bundle. If many replicates are to be seeded, a large number of asks and high volume of cell culture medium may be necessary depending on the yield for each cell line. In the case of human myoblasts, cells are sometimes larger than other common cell lines, which can make it more di cult to obtain high yields of cells while keeping con uence under 70%.
Make sure to become familiarized with the culture time and passage dilutions required for your speci c muscle cell line, as well as usual yields of cells per T75 or T175 ask at ~70% con uence. This is necessary to ensure that the required number of cells will be available at the day of the experiment. For example, in our case, we obtain approximately 6·10 6 cells in one T175 ask. Therefore, we make sure to have at least one T175 at 70% con uence for every two replicates on the day of the experiment. We perform passages at a 1:5-1:10 dilution. 11 On the day of seeding, sterilize the necessary amount of Petri dishes according to the following steps.
TIP: To the desired number of replicates, add at least two more Petri dishes for sterilization, which can be used as replacements in case one of the VELCRO ® pieces becomes detached during the sterilization process (this occurs in less than 10% of plates).
11.1 Place the Petri dishes on larger (150 mm Ø or similar) Petri dishes, for ease of manipulation ( Figure 4). Add 70% ethanol to each dish, up to the brim, and leave  11.3 After UV sterilization, turn the hood back on, and remove the PBS by aspiration with a glass Pasteur pipette and vacuum. Make sure to aspirate all of the liquid from the VELCRO ® anchors, which are quite absorbent. Then, leave the plates in the incubator for approximately 01:00:00 to dry the plates completely. This hydrophobic coating will facilitate detachment of the muscle bundles from the Petri dish. In order to coat only the middle region where the muscle bundle will be placed, and avoid soaking the velcro with a hydrophobic material, the Pluronic ® solution is applied with a sterile ne paintbrush. Sterilize the paintbrush by immersion in 70% ethanol for 00:15:00 , and dry by aspiration with vacuum. Ideally, choose a paintbrush with short and square bristles.
12.1 Apply Pluronic ® solution according to Figure 5. Ensure that the region is completely covered, by dipping the paintbrush into the solution and reapplying several times if needed. 12.2 Once all plates are coated, leave the dishes in the incubator ( 37 °C and 5 % (v/v) CO 2 ) for 01:00:00 . Importantly, during this time, the coating solution will completely evaporate, leaving only the solute on the surface. After this, it is not necessary to aspirate or wash the coating solution; the hydrogel is directly seeded onto the plates. In this way, the plates will remain completely dry at the time of seeding, which will enable correct spreading of the hydrogel as shown in Step 14.5.
13 Trypsinize cells for seeding according to standard procedures. A large volume of cell suspension may be obtained depending on the number of devices to be seeded, so for comfort, it can be collected in one of the used asks before counting. From there, take an aliquot of the cell suspension and count the cells.
Then, calculate the volume needed for obtaining 3·10 6 cells, and place this volume into individual Falcon tubes. Each Falcon tube will contain enough cells for one muscle bundle.
Centrifuge cells at 800 rpm for 00:05:00 . Seeding and differentiation of muscle bundles 14 After centrifuging cells, place the Falcon tubes in the hood and use them sequentially to make muscle bundles. The cells will be resuspended in a hydrogel containing the components listed in Figure 6. All components must be kept on ice before and during the seeding procedure. 14.2 Then, add the indicated amount of Matrigel ® ( 120 µL ) and homogenize the mixture gently by pipetting.
14.3 After this step, prepare a P1000 pipette with a pipette tip and set it to 300 µL . Set it aside until needed for seeding.
14.4 Add 30 µL of thrombin solution to the tube, and then swiftly homogenize the contents of the tube using the P1000 pipette (4-5 times, gently). Aspirate all of the volume and take it to one of the Petri dishes. 14.5 For seeding the hydrogel-cell mixture, quickly but carefully follow the directions in Figure 7. It is important to follow this order for distributing the liquid onto the VELCRO ® and forming the correct shape.
1. First, add part of the solution to both VELCRO ® anchors, and make sure to push the liquid into the loops by repeatedly pressing with the pipette tip in all directions. Due to the viscosity of the solution, it will not enter into the loops unless pressed into them, and this is important for forming a continuous structure with the middle portion. 2. Then, add most of the remaining solution to the middle part, and connect the two VELCRO ® anchors to each other. 3. Finally, distribute the hydrogel around the anchors to ensure that all sides are covered.
After seeding, the resulting structure will look at and wide ( Figure 8), as the hydrogel is still attached to the plate. Nevertheless, the shape will improve upon differentiation and compaction of the muscle bundles.
1. Figure 7. Procedure for correctly distributing the hydrogel solution onto the VELCRO ® anchors. 15 After two days in maintenance medium, switch the cells to differentiation medium. In Figure 9, the composition of differentiation medium for AB1079 cells is shown. This medium is prepared using a 3:1 mixture of DMEM and M199, respectively. Agrin is used for mimicking neural signaling for AChR clustering. 6-Aminocaproic acid (6-ACA) is used as an anti brinolytic agent, which aids in the long-term maintenance of the structure.  To prevent evaporation, keep muscle construct dishes inside a larger Petri dish (with lid) containing a small dish lled with sterile water.

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Validation of muscle differentiation Check that the muscle bundles detach from the plate within the rst 1-3 days of culture. When seen from the side, the muscle bundles should be slightly lifted from the Petri dish as shown in Figure 10. 17 During differentiation, take bright eld images of the muscle bundles at 4X and 10X magni cation. With these images, perform a directionality analysis to show alignment of cells. This can be done in ImageJ with the Directionality plugin, which will provide a Fast Fourier Transform (FFT) of the image, and a directionality histogram ( Figure 11). 18 In order to con rm that differentiation has taken place, perform immunostaining of three key markers: Sarcomeric α-actinin (SAA), Myosin Heavy Chain (MHC) and acetylcholine receptors (AChR). The rst two markers are needed to con rm the formation of sarcomeres (banding pattern), with SAA being more speci c for late-stage differentiation. The latter is relevant if it is desired to perform chemical stimulation with acetylcholine. After staining, perform confocal imaging of the samples. It is recommended to view all of these markers at 40X magni cation.   Incubate the blocking solution with gentle shaking.

18.3
In order to reduce the necessary amount of antibody solutions and enable mounting, cut the muscle constructs using a scalpel as shown in Figure 15. Pick up the cut-out middle portions using a ne paintbrush, and place them in 1h 40m 6h the well of a 24-well plate. Note that the plate and well must contain PBS, in order to facilitate detachment of the hydrogel from the paintbrush. Antibodies:

SAA:
Monoclonal Anti-α-Actinin (Sarcomeric) antibody produced in mouse Sigma Aldrich Catalog #A7811 18.5 In order to mount the samples for confocal imaging, it is necessary to use coverslips with small drops of nail polish at the corners (prepare in advance and let them dry). The height of the dried nail polish will create a gap for the muscle bundle to be mounted without risk of excessive deformation (Figure 16).
Transfer the muscle bundles onto a glass slide using a ne paintbrush, and cover them with Mowiol® 4-88 Sigma Aldrich Catalog #81381-50G mounting medium. Then, place the modi ed coverslip on top of the constructs (without applying pressure) and let the mounting medium harden for 1-2 days at 4 °C . 19 After 14 days in differentiation medium, cells can be stimulated electrically to prove that they have the ability to contract synchronically in response to electrical pulses. Contractions may be imaged in bright eld and processed using the MUSCLEMOTION plugin for ImageJ, or alternatively, calcium imaging can be performed using Fluo-4 AM and subsequent ΔF/F 0 analysis.
19.1 Before stimulation, it is necessary to build a simple stimulation device, which consists of two graphite electrodes attached to the lid of a 35 mm ∅ Petri dish ( Figure 17). The graphite rods are bound to two copper wires, which go through two small holes on the lid of the Petri dish. The holes and wires are sealed together with Loctite ® Super glue.

Electrical pulse stimulation
The electrical stimulation setup consists of this stimulation device, an electrical pulse generator (we use Aim TTi TG2512A), and an oscilloscope. The pulse generator is connected to a T-adapter, which connects to the oscilloscope and to two positive and negative cables with alligator clips. The alligator clips are clipped to the wires on the electrodes.
For additional details on the setup of the electrical stimulation equipment, see Figure S7 in the associated PLOS One article.  19.3 Inside the biological safety hood, place the electrode lid onto the Petri dish of interest, with the electrodes parallel to the muscle bundle ( Figure 17). Take the closed plate to the microscope, and fasten it to the microscope stage using appropriate holder, or alternatively, two pieces of tape placed at the sides. This will avoid accidental opening of the lid when attaching the electrodes to the pulse generator.
For additional details on the setup of the samples for electrical stimulation, see Figure S7 in the associated PLOS One article.
19.4 For stimulation, the electrodes on the lid are connected to a pulse generator, programmed with the following recommended parameters: Pulse duration: 10 ms Frequency: 1 Hz Amplitude: 10 V (monophasic) Higher frequencies may be tested if it is desired to study the tetanization of the muscle ber. Different voltages (5-20V) may be required depending on the cell line in order to see a robust contraction response. Pulse durations between 2-10 ms may also be tested. 30m The pulse generator is connected to an oscilloscope to verify that the pulses are correctly generated.
19.5 Program the microscope settings. Depending on the type of analysis that has been chosen (contraction analysis or calcium transient analysis), the microscope settings will be programmed differently. Go to Step 33 or Step 41, respectively.
19.6 Attach the graphite electrode cables to the pulse generator using alligator clips, and fasten these cables to the microscope stage using a piece of tape, to avoid unwanted movement of the cables.
19.7 Focus on the muscle bundle at 20X magni cation, in bright eld. It is recommended to focus on the edge of the bundle, as shown in Figure 18, to obtain better contraction images.  It is best to prepare the acetylcholine solution fresh, on the day that stimulation will be performed, to avoid degradation.
22 Before stimulation with chemical solutions, ensure that the volume inside the Petri dish is 3 ml.
If it is lower, change/add medium before stimulation. Medium may have experienced evaporation in the incubator, which can affect the nal concentration of the added solutions.

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Turn on the microscope. Make sure to place both chemical solutions (ACh and KCl) next to the microscope. Bring pipettes and pipette tips in order to add the chemical solutions when needed.
If the plate contains 3 ml of medium, the amounts to be added of each solution are: ACh: 1:100 dilution --> 30 μl --> P100 pipette KCl: 1:10 dilution --> 300 μl --> P1000 pipette 24 Program the microscope settings. Depending on the type of analysis that has been chosen (contraction analysis or calcium transient analysis), the microscope settings will be programmed differently. Go to Step 33 or Step 41, respectively.
Place the plate at the uorescence microscope, and focus on the muscle bundle at 20X magni cation. It is recommended to focus on the exterior part of the bundle (as in Figure 18) which will be more exposed to the chemical stimulant.

Chemical stimulation with acetylcholine and KCl
Focus with the lid taken off of the plate, to allow for quick addition of stimulation solutions. This means that the plate will no longer be sterile after stimulation.
26 Start the video recording and perform the stimulation according to the chosen stimulation regimen. In our experience, the stimulation regimens shown in Figure 20 and Figure 21 have been appropriate for quantifying the response to the applied stimulation, but may be modi ed for different needs.
At the chosen times, add the chemical solutions to the medium next to the recorded region, making sure to avoid disturbing the image. For this, pipette quickly (accurate start time of stimulation) but steadily (minimize vibrations in the medium and muscle construct).  27 For optical stimulation, it is necessary to use a myoblast cell line which has previously been made to express channelrhodopsin-2 (ChR2). We have generated ChR2+ human myoblasts by viral infection and FACS puri cation, as described in a previous publication from our group:

Optical stimulation of ChR2+ cells
After 14 days in differentiation medium, cells expressing ChR2 can be optically stimulated.
Contractions are imaged in bright eld and processed using the MUSCLEMOTION plugin for ImageJ.

Neuromuscular Activity Induces Paracrine Signaling and Triggers Axonal Regrowth after Injury in
Micro uidic Lab-On-Chip Devices.. Cells.
https://doi.org/pii:E302.10.3390/cells9020302 28 The optical stimulation setup consists of a coolLED pE-300 illumination system coupled to an inverted microscope (i.e., Olympus IX71) to visualize the muscle contraction. The TTL control of the illumination system allows for the delivery of precisely timed sequences of light. The TTL can be directed to an Arduino-UNO™ microcontroller pulse generator or alternatively a PulserPlus generator and Pulser v3.1 software (Prizmatix, Israel). The average light intensity with this setup is ≈ 20-25 W/cm 2 , measured at the culture dish with a Newport 1919 optical power meter (Newport Photonics, USA).
The two alternative setups are shown in Figure 22 and Figure 23, respectively.  30 Program the microscope software to record a bright eld video with the parameters described in Step 33. Then, turn off the pulse generator and return the cells to the incubator. Repeat the process with any remaining plates.
If performing contraction analysis, program the following recommended microscope settings for bright eld videos: Resolution: 1024x1024 px, 16-bit Total time of recording: 1 min or more, depending on chosen stimulation regimen Frame speed: 40 fps (1 image every 25 ms) Exposure time: lower than 25 ms, check that the image acquisition time is faster than the desired frame speed.
Regarding software, commercial (such as Olympus cellSens) or open-source software (μManager) can be used to program these parameters. 34 After bright eld imaging of contraction, analyze the contraction response with the MUSCLEMOTION plugin for ImageJ. This protocol can be used for electrical, chemical or optical stimulation videos. 34.1 If the videos are obtained in certain microscope software formats such as .vsi, it will be necessary to open them with the Bioformats plugin (Bioformats Importer), and then save them as a .tif "Image Sequence". Opening and converting large videos may be time-consuming depending on your computer's processing speed.
34.2 Open the MuscleMotion plugin in ImageJ and click "Run". Select the frame rate according to the frame rate in the input video. Leave all other parameters as shown in Figure 25 and click OK. This will open a le selection menu to select the output directory (recommended to create a new folder) and the input directory (the folder with the TIFF image sequence). 34.3 After running the program, the processing may take several minutes to be completed. The les that will be obtained in the output folder are:

Contraction analysis with MUSCLEMOTION
Reference frame Contraction: graph and text le Contraction speed: graph and text le The text les contain contraction or contraction speed information at each frame, which can be used for creating a graph in any other software of your choice (for example, GraphPad Prism, as in Figure 26). Fluo-4 cannot be used as a calcium indicator in combination with ChR2 + cell lines, as both are excited with the same wavelength (blue light). If it is desired to combine calcium imaging with optogenetic stimulation, a genetically encoded calcium indicator with red emission spectrum (RCaMP) may be combined with ChR2. In this way, cells are optically stimulated with blue light and calcium imaging is performed using a red light lter. Using an LED source is more appropriate for performing calcium imaging, as the light intensity remains more stable during recording than it is when using a halogen lamp.
42 In order to analyze videos obtained from Fluo-4 AM calcium imaging experiments, we use ImageJ for processing and obtaining ΔF/F 0 data. In order to analyze calcium transients, individual cells are manually delimited using the ImageJ ROI manager. Then, uorescence intensity data is extracted at each timepoint. This data is normalized to the mean basal uorescence. 1h 30m

Calcium transient analysis
As an example, a video using chemical stimulation with 100 micromolar (µM) ACh and 100 millimolar (mM) KCl is analyzed in the following steps. This video was recorded at 100 ms/frame (10 fps), for a total of 4 minutes.
43 First, open the video le in ImageJ. Play the video le using the sliding bar, and identify the timepoint or frame at which the stimulation was initiated.
For example, in the case of a video with the stimulation regimen shown in Figure 21 (with addition of acetylcholine and KCl), key timepoints would be 1 min (60000 ms = 600 frames) for ACh addition and 3 min (180000 ms = 1800 frames) for KCl addition, respectively.
By moving the sliding bar back and forth, visually identify cells that respond to the stimulation after the start of stimulation (see differences between Figure 27 and Figure 28). These cells will later be selected as regions of interest (ROIs), as explained in the following steps.   44.2 Using the polygon selection tool , outline each of the cells of interest as in Figure 29. After closing each outline, on the ROI manager, click on "Add" before outlining the next cell. To see all the ROIs, click on "Show all" At the end of the selection, the ROI set can be saved using More >> Save. 44.3 Once the ROIs are selected and saved, go to Analyze > Set Measurements and select "Mean gray value" only ( Figure 30). In this way, only the uorescence information will be extracted from the ROIs. Click OK. 44.4 Then, using the ROI manager, click on More >> Multi Measure to measure mean gray value of all ROIs at each timepoint ( Figure 31). Enable the rst two options on the window that pops up ( Figure 32).  44.5 After the measurements are completed, the results will pop up in a Results window. Each column contains the uorescence intensity data for one ROI at all the timepoints (each row is one frame). Save these results and/or copy them to an Excel le (or other similar spreadsheet software). Select the projection type, which is "Average Intensity", and click OK.  45.2 After this, a Z projection of the video will pop up. In this image, select a ROI containing only the background, which can be any shape (in Figure 36, an ellipse was used). 45.3 While this ROI is selected, go to Analyze > Measure in order to obtain the uorescence value for this ROI. Save the result that pops up (this will be F 0 ). 45.4 Using Excel or another spreadsheet software of your choice, normalize the uorescence data from each ROI to the F 0 as shown in Figure 37. 46 Use a software of your choice to generate an XY plot of the ΔF/F 0 over time. In our case, we use GraphPad Prism software, but other open-source software such as Veusz can be used instead.
X values: time, in increments of 100 ms, or as many ms as separate each frame. Y values: ΔF/F 0 of each ROI (plot all ROIs in the same graph).
In order to visualize the point at which the stimulation was applied, arrows can be added as annotations. It is also helpful to show each ROI in a different line color.